Carotenoids are widely distributed natural pigments that are responsible for many of the yellow, orange and red colors seen in living organisms. They have important commercial uses as coloring agents in the food industry, as feed and food additives, in cosmetics and as provitamin A precursors.
The plant species Adonis aestivalis produces flowers with petals that are deep red in color and nearly black at the base of the petals due to the accumulation of ketocarotenoid and other carotenoid pigments (Neamtu et al., Rev. Roum. Biochim. 6:157, 1969). This pattern of carotenoid accumulation accounts for the common name of some varieties of this species: summer pheasant""s eye.
Among the carotenoids identified in the petals of the red petal varieties of these various species is the ketocarotenoid astaxanthin (3,3xe2x80x2-dihydroxy-4,4xe2x80x2-diketo-b,b-carotene; see FIG. 1). Various other ketocarotenoids (see FIG. 1) including 3-hydroxyechinenone (3-hydroxy-4-keto-b,b-carotene),adonirubin (3-hydroxy-4,4xe2x80x2-diketo-b,b-carotene) adonixanthin (3,3xe2x80x2-dihydroxy-4-keto-b,b-carotene) and isozeaxanthin (4,4xe2x80x2-dihydroxy-b,b-carotene; see T. W. Goodwin, The Biochemistry of the Carotenoids, vol I. Plants, 2nd edition, 1980, page 147) have also been reported. The latter compound is consistent with speculation that the 4-hydroxy may be an intermediate in the formation of the 4-keto group.
There is appreciable interest in the biological production of carotenoids, in particular the orange-colored ketocarotenoids such as astaxanthin and canthaxanthin (FIG. 1), and in the modification of carotenoid composition. For this reason, an A. aestivalis flower cDNA library was constructed and screened for cDNAs encoding enzymes (hereinafter referred to as xe2x80x9cketolasesxe2x80x9d although the specific biochemical activity has not yet been established) involved in the conversion of b-carotene into orange compounds with absorption properties similar to those exhibited by common ketocarotenoids such as canthaxanthin (FIG. 1). Two distinctly different Adonis aestivalis cDNAs were obtained from among a number of cDNAs that were selected on this basis.
Thus, a first aspect of the present invention is a purified nucleic acid sequence which encodes for a protein having ketolase enzyme activity and has the nucleic acid sequence of SEQ ID NO: 1 or 3.
The invention also includes a purified nucleic acid sequence which encodes for a protein having ketolase enzyme activity and having the amino acid sequence of SEQ ID NO: 2 or 4.
The invention also includes vectors which comprise any portion of the nucleic acid sequences listed above, and host cells transformed with such vectors.
Another aspect of the present invention is a method of producing a ketocarotenoid in a host cell, the method comprising
inserting into the host cell a vector comprising a heterologous nucleic acid sequence which encodes for a protein having ketolase enzyme activity and comprises (1) SEQ ID NO: 1 or 3 or (2) a sequence which encodes the amino acid sequence of SEQ ID NO: 2 or 4, wherein the heterologous nucleic acid sequence is operably linked to a promoter; and
expressing the heterologous nucleic acid sequence, thereby producing the ketolase enzyme.
Another subject of the present invention is a method of modifying the production of carotenoids in a host cell, relative to an untransformed host cell, the method comprising
inserting into a host cell which already produces carotenoids a vector comprising a heterologous nucleic acid sequence which encodes for a protein having ketolase enzyme activity and comprises (1) SEQ ID NO: 1 or 3 or (2) a sequence which encodes the amino acid sequence of SEQ ID NO: 2 or 4, wherein the heterologous nucleic acid sequence is operably linked to a promoter; and
expressing the heterologous nucleic acid sequence in the host cell to modify the production of the carotenoids in the host cell, relative to an untransformed host cell.